This proposal utilizes the isolated perfused rabbit heart model of global ischemia to characterize the molecular events leading to cell death during ischemia and reperfusion. Biochemical and histological analysis are coupled to cell-free assays designed to evaluate protein-protein interactions. Myocardial ischemia is accompanied by ionic shifts, free radical production, and a commitment to cell death by apoptosis and necrosis. Proteolysis is one mechanism to irreversibly commit the cell to a death program. Bid is a member of the pro-apoptotic BH3-only subset of the Bc1-2 family. Bid is activated by proteolysis to translocate to the mitochondria to direct cytochrome c release. Loss of cytochrome c impairs mitochondrial respiration, may favor superoxide production, and may contribute to delayed caspase activation. Bid can be cleaved and activated by caspase-8, calpain, and lysosomal proteases. We have evidence that Bid is cleaved by calpain during myocardial ischemia, and propose to determine whether this is of physiological significance. ARC (Apoptosis Repressor with CARD Domain) is an anti-apoptotic molecule expressed at high levels in the heart. ARC has been shown to inhibit caspase-8 and prevent cytochrome c release; we find that ARC prevents cytochrome c release mediated by cleaved active Bid, suggesting a direct interaction. We propose to examine the interaction between ARC and Bid. We also find that ARC is susceptible to proteolysis during ischemia, and find that calpain is one protease that is able to cleave and inactivate ARC. We propose to examine the physiological significance of ARC proteolysis in ischemia. In order to establish more useful tools for studying the molecular events in ischemia, we propose to develop the methodology for TAT protein transduction in the isolated heart model.